Preclinical validation of an Escherichia coli O-antigen glycoconjugate for the prevention of serotype O1 invasive disease.

A US collection of invasive Escherichia coli serotype O1 bloodstream infection (BSI) isolates were assessed for genotypic and phenotypic diversity as the basis for designing a broadly protective O-antigen vaccine. Eighty percent of the BSI isolate serotype O1 strains were genotypically ST95 O1:K1:H7. The carbohydrate repeat unit structure of the O1a subtype was conserved in the three strains tested representing core genome multi-locus sequence types (MLST) sequence types ST95, ST38, and ST59. A long-chain O1a CRM197 lattice glycoconjugate antigen was generated using oxidized polysaccharide and reductive amination chemistry. Two ST95 strains were investigated for use in opsonophagocytic assays (OPA) with immune sera from vaccinated animals and in murine lethal challenge models. Both strains were susceptible to OPA killing with O1a glycoconjugate post-immune sera. One of these, a neonatal sepsis strain, was found to be highly lethal in the murine challenge model for which virulence was shown to be dependent on the presence of the K1 capsule. Mice immunized with the O1a glycoconjugate were protected from challenges with this strain or a second, genotypically related, and similarly virulent neonatal isolate. This long-chain O1a CRM197 lattice glycoconjugate shows promise as a component of a multi-valent vaccine to prevent invasive E. coli infections. IMPORTANCE: The Escherichia coli serotype O1 O-antigen serogroup is a common cause of invasive bloodstream infections (BSI) in populations at risk such as newborns and the elderly. Sequencing of US BSI isolates and structural analysis of O polysaccharide antigens purified from strains that are representative of genotypic sub-groups confirmed the relevance of the O1a subtype as a vaccine antigen. O polysaccharide was purified from a strain engineered to produce long-chain O1a O-antigen and was chemically conjugated to CRM197 carrier protein. The resulting glycoconjugate elicited functional antibodies and was protective in mice against lethal challenges with virulent K1-encapsulated O1a isolates.

Vibrational properties of disordered stealthy hyperuniform 1D atomic chains.

Disorder hyperuniformity is a recently discovered exotic state of many-body systems that possess a hidden order in between that of a perfect crystal and a completely disordered system. Recently, this novel disordered state has been observed in a number of quantum materials including amorphous 2D graphene and silica, which are endowed with unexpected electronic transport properties. Here, we numerically investigate 1D atomic chain models, including perfect crystalline, disordered stealthy hyperuniform (SHU) as well as randomly perturbed atom packing configurations to obtain a quantitative understanding of how the unique SHU disorder affects the vibrational properties of these low-dimensional materials. We find that the disordered SHU chains possess lower cohesive energies compared to the randomly perturbed chains, implying their potential reliability in experiments. Our inverse partition ratio (IPR) calculations indicate that the SHU chains can support fully delocalized states just like perfect crystalline chains over a wide range of frequencies, i.e.ω∈(0,100)cm-1, suggesting superior phonon transport behaviors within these frequencies, which was traditionally considered impossible in disordered systems. Interestingly, we observe the emergence of a group of highly localized states associated withω∼200cm-1, which is characterized by a significant peak in the IPR and a peak in phonon density of states at the corresponding frequency, and is potentially useful for decoupling electron and phonon degrees of freedom. These unique properties of disordered SHU chains have implications in the design and engineering of novel quantum materials for thermal and phononic applications.

Computational design of anisotropic stealthy hyperuniform composites with engineered directional scattering properties.

Disordered hyperuniform materials are an emerging class of exotic amorphous states of matter that endow them with singular physical properties, including large isotropic photonic band gaps, superior resistance to fracture, and nearly optimal electrical and thermal transport properties, to name but a few. Here we generalize the Fourier-space-based numerical construction procedure for designing and generating digital realizations of isotropic disordered hyperuniform two-phase heterogeneous materials (i.e., composites) developed by Chen and Torquato [Acta Mater. 142, 152 (2018)1359-645410.1016/j.actamat.2017.09.053] to anisotropic microstructures with targeted spectral densities. Our generalized construction procedure explicitly incorporates the vector-dependent spectral density function χ[over ̃]_{_{V}}(k) of arbitrary form that is realizable. We demonstrate the utility of the procedure by generating a wide spectrum of anisotropic stealthy hyperuniform microstructures with χ[over ̃]_{_{V}}(k)=0 for k∈Ω, i.e., complete suppression of scattering in an "exclusion" region Ω around the origin in Fourier space. We show how different exclusion-region shapes with various discrete symmetries, including circular-disk, elliptical-disk, square, rectangular, butterfly-shaped, and lemniscate-shaped regions of varying size, affect the resulting statistically anisotropic microstructures as a function of the phase volume fraction. The latter two cases of Ω lead to directionally hyperuniform composites, which are stealthy hyperuniform only along certain directions and are nonhyperuniform along others. We find that while the circular-disk exclusion regions give rise to isotropic hyperuniform composite microstructures, the directional hyperuniform behaviors imposed by the shape asymmetry (or anisotropy) of certain exclusion regions give rise to distinct anisotropic structures and degree of uniformity in the distribution of the phases on intermediate and large length scales along different directions. Moreover, while the anisotropic exclusion regions impose strong constraints on the global symmetry of the resulting media, they can still possess structures at a local level that are nearly isotropic. Both the isotropic and anisotropic hyperuniform microstructures associated with the elliptical-disk, square, and rectangular Ω possess phase-inversion symmetry over certain range of volume fractions and a percolation threshold ϕ_{c}≈0.5. On the other hand, the directionally hyperuniform microstructures associated with the butterfly-shaped and lemniscate-shaped Ω do not possess phase-inversion symmetry and percolate along certain directions at much lower volume fractions. We also apply our general procedure to construct stealthy nonhyperuniform systems. Our construction algorithm enables one to control the statistical anisotropy of composite microstructures via the shape, size, and symmetries of Ω, which is crucial to engineering directional optical, transport, and mechanical properties of two-phase composite media.

Preclinical Immunogenicity and Efficacy of Optimized O25b O-Antigen Glycoconjugates To Prevent MDR ST131 E. coli Infections.

Multivalent O-antigen polysaccharide glycoconjugate vaccines are under development to prevent invasive infections caused by pathogenic Enterobacteriaceae. Sequence type 131 (ST131) Escherichia coli of serotype O25b has emerged as the predominant lineage causing invasive multidrug-resistant extraintestinal pathogenic E. coli (ExPEC) infections. We observed the prevalence of E. coli O25b ST131 among a contemporary collection of isolates from U.S. bloodstream infections from 2013 to 2016 (n = 444) and global urinary tract infections from 2014 to 2017 (n = 102) to be 25% and 24%, respectively. To maximize immunogenicity of the serotype O25b O antigen, we investigated glycoconjugate properties, including CRM197 carrier protein cross-linking (single-end versus cross-linked "lattice") and conjugation chemistry (reductive amination chemistry in dimethyl sulfoxide [RAC/DMSO] versus ((2-((2-oxoethyl)thio)ethyl)carbamate [eTEC] linker). Using opsonophagocytic assays (OPAs) to measure serum functional antibody responses to vaccination, we observed that higher-molecular-mass O25b long-chain lattice conjugates showed improved immunogenicity in mice compared with long- or short-chain O antigens conjugated via single-end attachment. The lattice conjugates protected mice from lethal challenge with acapsular O25b ST131 strains as well as against hypervirulent O25b isolates expressing K5 or K100 capsular polysaccharides. A single 1-µg dose of long-chain O25b lattice conjugate constructed with both chemistries also elicited robust serum IgG and OPA responses in cynomolgus macaques. Our findings show that key properties of the O-antigen carrier protein conjugate such as saccharide epitope density and degree of intermolecular cross-linking can significantly enhance functional immunogenicity.

Demonstration of the preclinical correlate of protection for Staphylococcus aureus clumping factor A in a murine model of infection.

The Staphylococcus aureus virulence factor clumping factor A (ClfA) is a component of an investigational S. aureus prophylactic vaccine. ClfA enables S. aureus to bind to fibrinogen and platelets during the initial stages of invasive disease. Here we demonstrate that ectopic expression of ClfA is sufficient to render nonpathogenic Lactococcus lactis lethal in a murine model of systemic infection. In contrast, L. lactis expressing ClfAY338A, which cannot bind fibrinogen, did not cause death in the mice. Pathogenicity was also prevented by immunization with ClfA. This model was then used to define a preclinical correlate of protection by measuring functional antibody in a S. aureus fibrinogen binding inhibition assay (FBI) and correlating that titer with protective outcomes. Although many humans have pre-existing antibodies that bind to ClfA, only sera with a threshold functional titer in the FBI were protective in this preclinical model. This confirms that fibrinogen binding is critical for ClfA-mediated pathogenesis and demonstrates that functional antibodies against ClfA are sufficient to protect against ClfA-mediated pathogenesis in vivo, enabling the definition of a preclinical correlate of protection for ClfA-containing vaccines based on FBI titer.

Characterization and sequence analysis of extended-spectrum-{beta}-lactamase-encoding genes from Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates collected during tigecycline phase 3 clinical trials.

In concert with the development of novel beta-lactams and broad-spectrum cephalosporins, bacterially encoded beta-lactamases have evolved to accommodate the new agents. This study was designed to identify, at the sequence level, the genes responsible for the extended-spectrum-beta-lactamase (ESBL) phenotypes of Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates collected during the global tigecycline phase 3 clinical trials. PCR assays were developed to identify and clone the bla(TEM), bla(SHV), bla(OXA), and bla(CTX) genes from clinical strains. Isolates were also screened for AmpC genes of the bla(CMY), bla(ACT), bla(FOX), and bla(DHA) families as well as the bla(KPC) genes encoding class A carbapenemases. E. coli, K. pneumoniae, and P. mirabilis isolates with ceftazidime MICs of > or =2 microg/ml were designated possible ESBL-producing pathogens and were then subjected to a confirmatory test for ESBLs by use of Etest. Of 272 unique patient isolates, 239 were confirmed by PCR and sequencing to carry the genes for at least one ESBL, with 44% of the positive isolates harboring the genes for multiple ESBLs. In agreement with current trends for ESBL distribution, bla(CTX-M)-type beta-lactamase genes were found in 83% and 71% of the ESBL-positive E. coli and K. pneumoniae isolates, respectively, whereas bla(SHV) genes were found in 41% and 28% of the ESBL-positive K. pneumoniae and E. coli isolates, respectively. Ninety-seven percent of the E. coli and K. pneumoniae isolates were tigecycline susceptible (MIC(90) = 2 microg/ml), warranting further studies to define the therapeutic utility of tigecycline against strains producing ESBLs in a clinical setting.

Microbial fouling of a reverse osmosis municipal water treatment system.

Microbial fouling of a municipal water treatment system using reverse osmosis was investigated. From a combination of growth and molecular assays, it was discovered that the prefilter unit concentrated and facilitated microbial growth, and such growth led to microbial fouling of the reverse osmosis unit. Few cells were observed in the prefilter influent, but substantial microbial contamination was observed in the prefilter effluent, and this correlated with increasing headloss across the prefilter. The effluent caused microbial fouling of the leading elements of the reverse osmosis unit, as determined by reduced permeate flow, analysis of the elements, and assays of the membrane foulant. Both the introduction of microorganisms to the reverse osmosis unit from the prefilter unit and headloss across the prefilter could be effectively controlled through cleansing of the prefilter housing unit with sulfuric acid. Such treatments must be performed at appropriate intervals to prevent subsequent microbial growth in the prefilter unit.

MarA-mediated overexpression of the AcrAB efflux pump results in decreased susceptibility to tigecycline in Escherichia coli.

OBJECTIVES: The purpose of this study was to characterize decreased susceptibility to tigecycline in clinical isolates of Escherichia coli obtained during Phase 3 clinical trials. METHODS: Gene expression was analysed by transcriptional profile analysis and RT-PCR. Transposon mutagenesis with IS903kan was used for selection of transposon mutants. Transposon insertions were mapped by DNA sequencing and PCR analyses. The MICs were determined by broth microdilution. RESULTS: Both transcriptional profile analysis and Taqman RT-PCR demonstrated increased expression levels of MarA, a transcriptional activator, and AcrAB, an RND-type efflux pump, in the strains with elevated tigecycline MICs. Transposon mutagenesis generated nine mutants, the majority of which had either marA or acrB inactivated. Sequence analysis revealed a single nucleotide insertion in the open reading frame of the marR gene in less-susceptible strains of E. coli. CONCLUSIONS: This study suggested that a loss of MarR functionality due to a frameshift mutation resulted in constitutive overproduction of MarA and AcrAB and, consequently, in decreased susceptibility to tigecycline in clinical isolates of E. coli.

RamA, a transcriptional regulator, and AcrAB, an RND-type efflux pump, are associated with decreased susceptibility to tigecycline in Enterobacter cloacae.

Tigecycline, a novel broad-spectrum glycylcycline antibiotic, is active against many gram-positive and gram-negative bacterial pathogens including most strains of Enterobacter cloacae. Recently, however, a few clinical strains of E. cloacae with decreased susceptibility to tigecycline were isolated. In this study, two tigecycline-susceptible mutants of E. cloacae, GC7696 and GC7697, were obtained by transposon mutagenesis of a tigecycline-resistant clinical isolate G946. Transposon insertions were mapped to either the acrA or acrB genes. Restoration of the original resistant phenotype occurred when GC7696 and GC7697 were transcomplemented with a plasmid harboring the intact acrAB region amplified from G946. Northern blot analysis of G946 and several other E. cloacae clinical strains that exhibited decreased susceptibility to tigecycline, revealed increased levels of the acrAB transcript. In addition, overexpression of acrAB correlated with increased expression of the ramA gene, whereas the expression of another transcriptional activator, marA, was not changed. These results suggest that decreased susceptibility to tigecycline in E. cloacae is the result of RamA-mediated overexpression of the AcrAB efflux pump.

AdeABC multidrug efflux pump is associated with decreased susceptibility to tigecycline in Acinetobacter calcoaceticus-Acinetobacter baumannii complex.

OBJECTIVES: To investigate the role of the AdeABC multidrug efflux pump in the decreased susceptibility of clinical isolates of Acinetobacter calcoaceticus-Acinetobacter baumannii complex to tigecycline. METHODS: Gene expression was analysed by Taqman RT-PCR. A single cross-over achieved insertional inactivation of the adeB gene with a suicide plasmid construct carrying an adeB fragment obtained by PCR. Analysis of the adeRS locus was performed by PCR and sequencing. Ribotyping was performed with the RiboPrinter system. MICs were determined by Etest. RESULTS: Expression analysis revealed constitutive overexpression of adeABC in less-susceptible clinical isolates G5139 and G5140 (tigecycline MIC=4 mg/L) when compared with the isogenic clinical isolates G4904 and G5141 (MIC=1.5 mg/L). Insertional mutants GC7945 (adeB knockout in G5139) and GC7951 (adeB knockout in G5140) were obtained, which resulted in tigecycline MICs of 0.5 mg/L. As reported previously, the expression of adeABC is regulated by the two-component signalling system encoded by the adeR and adeS genes. PCR and sequencing analyses revealed an insertion of an IS(ABA-1) element in the adeS gene of G5139 and G5140. CONCLUSIONS: The results of this study suggest that decreased susceptibility to tigecycline in the A. calcoaceticus-A. baumannii complex is associated with the overexpression of the AdeABC multidrug efflux pump.

Mixed pollutant degradation by Methylosinus trichosporium OB3b expressing either soluble or particulate methane monooxygenase: can the tortoise beat the hare?

Methanotrophs have been widely investigated for in situ bioremediation due to their ubiquity and their ability to degrade halogenated hydrocarbons through the activity of methane monooxygenase (MMO). It has been speculated that cells expressing the soluble form of MMO (sMMO) are more efficient in cleaning up sites polluted with halogenated hydrocarbons due to its broader substrate range and relatively fast degradation rates compared cells expressing the other form of MMO, the particulate MMO (pMMO). To examine this issue, the biodegradation of mixtures of chlorinated solvents, i.e., trichloroethylene (TCE), trans-dichloroethylene (t-DCE), and vinyl chloride (VC), by Methylosinus trichosporium OB3b in the presence of methane using either form of MMO was investigated over longer time frames than those commonly used, i.e., days instead of hours. Growth of M. trichosporium OB3b along with pollutant degradation were monitored and analyzed using a simple comparative model developed from the Omega model created for analysis of the competitive binding of oxygen and carbon dioxide by ribulose bisphosphate carboxylase. From these findings, it appears that at concentrations of VC, t-DCE, and TCE greater than 10 microM each, methanotrophs expressing pMMO have a competitive advantage over cells expressing sMMO due to higher growth rates. Despite such an apparent growth advantage, pMMO-expressing cells degraded less of these substrates at these concentrations than sMMO-expressing cells during active growth. If the concentrations were increased to 100 muM, however, not only did pMMO-expressing cells grow faster, they degraded more of these pollutants and did so in a shorter amount of time. These findings suggest that the relative rates of growth substrate and pollutant degradation are important factors in determining which form of MMO should be considered for pollutant degradation.

Pyrazolidine-3,5-diones and 5-hydroxy-1H-pyrazol-3(2H)-ones, inhibitors of UDP-N-acetylenolpyruvyl glucosamine reductase.

A series of pyrazolidine-3,5-dione and 5-hydroxy-1H-pyrazol-3(2H)-one inhibitors of Escherichia coli UDP-N-acetylenolpyruvyl glucosamine reductase (MurB) has been prepared. The 5-hydroxy-1H-pyrazol-3(2H)-ones show low micromolar IC(50) values versus E. coli MurB and submicromolar minimal inhibitory concentrations (MIC) against Staphylococcus aureus GC 1131, Enterococcus faecalis GC 2242, Streptococcus pneumoniae GC 1894, and E. coli GC 4560 imp, a strain with increased outer membrane permeability. None of these compounds show antimicrobial activity against Candida albicans, a marker of eukaryotic toxicity. Moreover, these compounds inhibit peptidoglycan biosynthesis, as assessed by measuring the amount of soluble peptidoglycan produced by Streptococcus epidermidis upon incubation with compounds. A partial least squares projection to latent structures analysis shows that improving MurB potency and MIC values correlate with increasing lipophilicity of the C-4 substituent of the 5-hydroxy-1H-pyrazol-3(2H)-one core. Docking studies using FLO and PharmDock produced several binding orientations for these molecules in the MurB active site.

3,5-dioxopyrazolidines, novel inhibitors of UDP-N- acetylenolpyruvylglucosamine reductase (MurB) with activity against gram-positive bacteria.

A series of 3,5-dioxopyrazolidines was identified as novel inhibitors of UDP-N-acetylenolpyruvylglucosamine reductase (MurB). Compounds 1 to 3, which are 1,2-bis(4-chlorophenyl)-3,5-dioxopyrazolidine-4-carboxamides, inhibited Escherichia coli MurB, Staphyloccocus aureus MurB, and E. coli MurA with 50% inhibitory concentrations (IC50s) in the range of 4.1 to 6.8 microM, 4.3 to 10.3 microM, and 6.8 to 29.4 microM, respectively. Compound 4, a C-4-unsubstituted 1,2-bis(3,4-dichlorophenyl)-3,5-dioxopyrazolidine, showed moderate inhibitory activity against E. coli MurB, S. aureus MurB, and E. coli MurC (IC50s, 24.5 to 35 microM). A fluorescence-binding assay indicated tight binding of compound 3 with E. coli MurB, giving a dissociation constant of 260 nM. Structural characterization of E. coli MurB was undertaken, and the crystal structure of a complex with compound 4 was obtained at 2.4 A resolution. The crystal structure indicated the binding of a compound at the active site of MurB and specific interactions with active-site residues and the bound flavin adenine dinucleotide cofactor. Peptidoglycan biosynthesis studies using a strain of Staphylococcus epidermidis revealed reduced peptidoglycan biosynthesis upon incubation with 3,5-dioxopyrazolidines, with IC50s of 0.39 to 11.1 microM. Antibacterial activity was observed for compounds 1 to 3 (MICs, 0.25 to 16 microg/ml) and 4 (MICs, 4 to 8 microg/ml) against gram-positive bacteria including methicillin-resistant S. aureus, vancomycin-resistant Enterococcus faecalis, and penicillin-resistant Streptococcus pneumoniae.

Pulvinones as bacterial cell wall biosynthesis inhibitors.

Pulvinones were synthesized (>180) in arrays and evaluated as inhibitors of early stage cell wall biosynthesis enzymes MurA-MurD. Several pulvinones inhibited Mur enzymes with IC(50)'s in the 1-10 microg/mL range and demonstrated antibacterial activity against Gram-positive bacteria including methicillin-resistant Staphyloccus aureus, vancomycin-resistant Enterococcus faecalis, and penicillin-resistant Streptococcus pneumoniae.

Concordant copy number and transcriptional activity of genes mapping to derivative chromosomes 8 during cellular immortalization in vitro.

Deletion, rearrangement, or amplification of sequences mapping to chromosome 8 are frequently observed in human prostate and other tumors. However, it is not clear whether these events alter the transcriptional activity of the affected genes. To examine this question, we have utilized oligonucleotide microarray technology and compared the transcriptional patterns of normal human prostate tissues and five immortalized cell lines carrying either two normal chromosomes 8 or one normal and one derivative chromosome 8. Comparison of the transcriptional profiles of the tissues and cell lines identified 125 differentially expressed transcripts specific to chromosome 8, with 46 transcripts mapping to 8p and 79 transcripts mapping to 8q. The majority of genes mapping to 8p (44/46, 96%) were transcriptionally down-regulated in cells hemizygous for 8p, whereas the majority of genes mapping to 8q (58/79, 73%) were up-regulated in cells carrying three copies of 8q. Moreover, hemizygous alleles on 8p exhibited sub-haploinsufficient transcript levels for several genes that could be induced to haploinsufficient levels under hypomethylating conditions, suggesting that epigenetic regulation is a common mechanism for gene silencing in cells deleted for one copy of 8p. The results of these studies clearly demonstrate that alterations of gene copy number and transcriptional activity are directly correlated in cell lines harboring derivative chromosomes 8, and that these events are commonly observed during cellular immortalization in vitro.

Degradation of trafficking-defective long QT syndrome type II mutant channels by the ubiquitin-proteasome pathway.

Mutations in the human ether-a-go-go-related gene (hERG) cause chromosome 7-linked long QT syndrome type II (LQT2). We have shown previously that LQT2 mutations lead to endoplasmic reticulum (ER) retention and rapid degradation of mutant hERG proteins. In this study we examined the role of the ubiquitin-proteasome pathway in the degradation of the LQT2 mutation Y611H. We showed that proteasome inhibitors N-acetyl-L-leucyl-L-leucyl-L-norleucinal and lactacystin but not lysosome inhibitor leupeptin inhibited the degradation of Y611H mutant channels. In addition, ER mannosidase I inhibitor kifunensine and down-regulation of EDEM (ER degradation-enhancing alpha-mannosidase-like protein) also suppressed the degradation of Y611H mutant channels. Proteasome inhibition but not mannosidase inhibition led to the accumulation of full-length hERG protein in the cytosol. The hERG protein accumulated in the cytosol was deglycosylated. Proteasome inhibition also resulted in the accumulation of polyubiquitinated hERG channels. These results suggest that the degradation of LQT2 mutant channels is mediated by the cytosolic proteasome in a process that involves mannose trimming, polyubiquitination, and deglycosylation of mutant channels.

Influence of transcriptional activator RamA on expression of multidrug efflux pump AcrAB and tigecycline susceptibility in Klebsiella pneumoniae.

Tigecycline is an expanded broad-spectrum antibacterial agent that is active against many clinically relevant species of bacterial pathogens, including Klebsiella pneumoniae. The majority of K. pneumoniae isolates are fully susceptible to tigecycline; however, a few strains that have decreased susceptibility have been isolated. One isolate, G340 (for which the tigecycline MIC is 4 microg/ml and which displays a multidrug resistance [MDR] phenotype), was selected for analysis of the mechanism for this decreased susceptibility by use of transposon mutagenesis with IS903phikan. A tigecycline-susceptible mutant of G340, GC7535, was obtained (tigecycline MIC, 0.25 microg/ml). Analysis of the transposon insertion mapped it to ramA, a gene that was previously identified to be involved in MDR in K. pneumoniae. For GC7535, the disruption of ramA led to a 16-fold decrease in the MIC of tigecycline and also a suppression of MDR. Trans-complementation with plasmid-borne ramA restored the original parental phenotype of decreased susceptibility to tigecycline. Northern blot analysis revealed a constitutive overexpression of ramA that correlated with an increased expression of the AcrAB transporter in G340 compared to that in tigecycline-susceptible strains. Laboratory mutants of K. pneumoniae with decreased susceptibility to tigecycline could be selected at a frequency of approximately 4 x 10(-8). These results suggest that ramA is associated with decreased tigecycline susceptibility in K. pneumoniae due to its role in the expression of the AcrAB multidrug efflux pump.

AcrAB efflux pump plays a role in decreased susceptibility to tigecycline in Morganella morganii.

Transposon mutagenesis of a clinical isolate of Morganella morganii, G1492 (tigecycline MIC of 4 microg/ml), yielded two insertion knockout mutants for which tigecycline MICs were 0.03 microg/ml. Transposon insertions mapped to acrA, which is constitutively overexpressed in G1492, suggesting a role of the AcrAB efflux pump in decreased susceptibility to tigecycline in M. morganii.

Defective assembly and trafficking of mutant HERG channels with C-terminal truncations in long QT syndrome.

Mutations in the human ether-a-go-go-related gene (HERG) cause long QT syndrome type 2 (LQT2). HERG encodes a voltage-gated potassium channel consisting of four subunits. Tetrameric assembly is required for the formation of functional HERG channels. In the present work, we studied the role of assembly in HERG channel dysfunction of LQT2 mutations Q725X and R1014X, both of which cause truncations of the C-terminus of HERG channels. When expressed in HEK293 cells, Q725X did not generate HERG current, while R1014X generated HERG current with markedly reduced amplitude. Western blot analysis showed that both mutations caused defective trafficking of HERG channel proteins. Using sucrose gradient centrifugation we showed that wild type HERG and R1014X formed a tetrameric structure, whereas Q725X was expressed as a monomer. When coexpressed with wild type HERG, R1014X, but not Q725X, caused dominant negative suppression of wild type HERG current. Coimmunoprecipitation experiments showed that the lack of dominant negative effect by Q725X was due to failure of mutant subunits to coassemble with wild type subunits. These results suggest that the Q725X mutation causes HERG channel dysfunction by disruption of tetrameric assembly of HERG channels. In contrast, the R1014X mutation is capable of forming tetrameric structure, and it causes HERG channel dysfunction by defective trafficking of the mutant protein.